This invention relates to novel phosphohalohydrins, to the process for the production thereof and to the use thereof for stimulating Txcex39xcex42 lymphocytes bearing TCR receptors with Vxcex39 and Vxcex42 variable regions.
In healthy individuals, the Txcex3xcex4 lymphocytes of primates (humans, monkeys) present in the peripheral bloodstream usually constitute from 1 to 5% of the lymphocytes in the blood and play a role in the immune system. It has been demonstrated that they recognize their antigenic ligands by direct interaction with the antigen without presentation by molecules of the MHC by a presenting cell. Txcex39xcex42 lymphocytes (sometimes also known as Txcex32xcex42 lymphocytes) are Txcex3xcex4 lymphocytes bearing TCR receptors with Vxcex39 and Vxcex42 variable regions. They constitute the majority of Txcex3xcex4 lymphocytes in human blood.
When activated, Txcex3xcex4 lymphocytes exercise a strong cytotoxic activity which is unrestrained by the MHC and is particularly effective in killing various types of cells, in particular pathogenic cells. These may be cells infected by viruses (xe2x80x9cxcex3xcex4 T cell activation or anergy during infections: the role of nonpeptidic TCR ligands and HLA class I moleculesxe2x80x9d Fabrizio POCCIA et al, Journal of Leukocyte Biology, 62, 1997, p. 1-5), or by other intracellular parasites, such as mycobacteria (xe2x80x9cThe antituberculous Mycobacterium bovis BCG Vaccine is an attenuated Mycobacterial producer of phosphorylated nonpeptidic Antigens for human xcex3xcex4 T cellsxe2x80x9d Patricia CONSTANT et al, Infection and Immunity, vol. 63, no. 12, December 1995, p. 4628-4633); or by protozoans (xe2x80x9cPlasmodium falciparum stimuli for human xcex3xcex4 T Cells are related to phosphorylated Antigens of mycobacteriaxe2x80x9d Charlotte BEHR et al, Infection and Immunity, Vol. 64, no. 8, 1996, p. 2892-2896). They may also be cancer cells (xe2x80x9cCD94/NKG2 inhibitory receptor complex modulates both antiviral and antitumoral responses of polyclonal phosphoantigen-reactive Vxcex39 Vxcex42 T lymphocytesxe2x80x9d Fabrizio POCCIA et al, Journal of Immunology, 159, p. 6009-6015; xe2x80x9cStimulation of xcex3xcex4 T cells by phosphoantigensxe2x80x9d Jean-Jacques FOURNIE, Marc BONNEVILLE, Res. Immunol., 66th FORUM IN IMMUNOLOGY, 147, p. 338-347).
It has been demonstrated that, in the event of a mycobacterial infection, human Txcex39xcex42 lymphocytes react to four natural, nonpeptidic molecules of a phosphorylated structure, known as phosphoantigens, which exhibit stimulation activity at a concentration of 1 to 5 nM (nanomolar) (WO-95/20673 and xe2x80x9cStimulation of human xcex3xcex4 T cells by nonpeptidic Mycobacterial ligandsxe2x80x9d Patricia CONSTANT et al, Science, 264, p. 267-270).
These natural antigens have not been completely identified. Certain authors have erroneously presented them as alkene derivatives of pyrophosphate, in particular isopentenyl pyrophosphate IPP (U.S. Pat. No. 5,639,653 and xe2x80x9cNatural and Synthetic nonpeptide antigens recognized by human xcex3xcex4 T cellsxe2x80x9d, Yoshimasa TANAKA et al, Nature, 375, 1995, p. 155-158). It has nonetheless now been demonstrated that none of these prenyl pyrophosphates is active at a concentration of nanomolar magnitude. The best results which have been obtained have been unable to demonstrate activity at below 3 xcexcM for IPP and below 0.3 xcexcM for dimethylallyl-UTP and 3-methyl-2-hexene pyrophosphate. The minimum active concentration of these compounds is thus, at best, of the order to 100 times higher than that of natural phosphoantigens.
With regard to IPP, it should in particular be noted that the most recent of the above-stated publications make the mistake of deducing the structure of the isopentenyl radical solely on the basis of mass spectrometry and the detection of a certain level of bioactivity. Indeed, apart from the fact that the compound analyzed in these publications was not purified and that a mass spectrum cannot identify uncharged species, it may be demonstrated that there are in fact several thousand different chemical structures which may have the same molecular weight and be a substituent of pyrophosphate in these molecules.
The fact that the minimum active concentration for IPP is much higher (some 1000 times higher) and that the intensity of the Txcex39xcex42 lymphocyte responses obtained is much weaker than that for natural phosphoantigens demonstrates that IPP is not one of these natural phosphoantigens (xe2x80x9cA novel nucleotide-containing antigen for human blood xcex3xcex4 T lymphocytesxe2x80x9d, Y. Poquet et al, Eur. J. Immunol. 1996, 26, p. 2344-2349). This is moreover confirmed by numerous other observations: IPP is not found in sufficient concentration in mycobacterial extracts which stimulate Txcex39xcex42 lymphocytes; according to xe2x80x9cHigh pH anion exchange chromatographic analysis of phosphorylated compounds: application to isolation and characterization of non peptide mycobacterial antigensxe2x80x9d, Y. Poquet et al, Anal. Biochem, 243 no. 1, 1996, p. 119-126, IPP does not have the same chromatographic (HPAEC) characteristics as natural phosphoantigens; IPP and other natural isoprenoids are produced by all living cells, but these do not stimulate Txcex39xcex42 lymphocytes.
Moreover, it is known that substances having bioactivity of the order of or greater than 1 xcexcM are only rarely compatible with the economic constraints of operation on an industrial scale. The synthetic phosphoantigens which have hitherto been proposed thus cannot be processed on an industrial scale under acceptable economic conditions.
Natural phosphoantigens, on the other hand, may only be produced in very small quantities (WO 95/20673) and, since their precise chemical structure has still not yet been established, they cannot be synthesized. Economic industrial scale processing is thus likewise out of the question, despite their demonstrated great therapeutic worth.
The object of the invention is accordingly to provide novel chemical compounds which activate Txcex39xcex42 lymphocytes at a minimum activation concentration of below 100 nM, in particular of the order of 1 nM.
A further object of the invention is to provide compounds which may be linked to a large number of organic groups, in particular to natural or synthetic peptide groups, so as to permit multifunctional compounds to be obtained.
A further object of the invention is to provide such compounds which may be synthesized simply, quantitatively and at low cost, i.e. in a manner compatible with the economic constraints of production on an industrial scale.
A further object of the invention is to provide an advantageous synthetic pathway for these compounds.
A further object of the invention is to provide a process for the production of the compounds according to the invention.
A further object of the invention is to suggest uses for the compounds according to the invention as a Txcex39xcex42 lymphocyte activator and in particular therapeutic uses of the compounds according to the invention.
The invention accordingly provides compounds comprising at least one phosphohalohydrin group of the formula: 
where X is a halogen selected from among iodine, bromine and chlorine,
R1 is selected from among xe2x80x94CH3 and xe2x80x94CH2xe2x80x94CH3,
Cat+ represents one or more organic or inorganic cation(s) (including the proton), which may be identical or different in the same compound, and n is an integer between 2 and 20.
A compound according to the invention may in particular comprise one or more phosphohalohydrin group(s) selected from among the esters of the following groups (IUPAC nomenclature) or among the compounds formed from these groups:
3-(halomethyl)-3-butanol-1-yl diphosphate, 3-(halomethyl)-3-pentanol-1-yl diphosphate, 4-(halomethyl)-4-pentanol-1-yl diphosphate, 4-(halo methyl)-4-hexanol-1-yl diphosphate, 5-(halomethyl)-5-hexanol-1-yl diphosphate, 5-(halomethyl)-4-heptanol-1-yl diphosphate, 6-(halomethyl)-6-heptanol-1-yl diphosphate, 6-(halomethyl)-6-octanol-1-yl diphosphate, 7-(halo methyl)-7-octanol-1-yl diphosphate, 7-(halomethyl)-7-nonanol-1-yl diphosphate, 8-(halomethyl)-8-nonanol-1-yl diphosphate, 8-(halomethyl)-8-decanol-1-yl diphosphate, 9-(halomethyl)-9-decanol-1-yl diphosphate, 9-(halomethyl)-9-undecanol-1-yl diphosphate, 10-(halomethyl)-10-undecanol-1-yl diphosphate, 10-(halomethyl)-10-dodecanol-1-yl diphosphate, 11-(halomethyl)-11-dodecanol-1-yl diphosphate, 11-(halomethyl)-11-tridecanol-1-yl diphosphate, 12-(halomethyl)-12-tridecanol-1-yl diphosphate, 12-(halomethyl)-12-tetradecanol-1-yl diphosphate, 13-(halomethyl)-13-tetradecanol-1-yl -diphosphate, 13-(halomethyl)-13-pentadecanol-1-yl diphosphate, 14-(halomethyl)-14-pentadecanol-1-yl diphosphate, 14-(halomethyl)-14-hexadecanol-1-yl diphosphate, 15-(halomethyl)-15-hexadecanol-1-yl diphosphate, 15-(halomethyl)-15-heptadecanol-1-yl diphosphate, 16-(halomethyl)-16-heptadecanol-1-yl diphosphate, 16-(halomethyl)-16-octadecanol-1-yl diphosphate, 17-(halomethyl)-17-octadecanol-1-yl diphosphate, 17-(halomethyl)-17-nonadecanol-1-yl diphosphate, 18-(halomethyl)-18-nonadecanol-1-yl diphosphate, 18-(halomethyl)-18-eicosanol-1-yl diphosphate, 19-(halomethyl)-19-eicosanol-1-yl diphosphate, 19-(halomethyl)-11-heneicosanol-1-yl diphosphate, 20-(halomethyl)-20-heneicosanol-1-yl diphosphate, 20-(halomethyl)-20-docosanol-1-yl diphosphate, 21-(halomethyl)-21-docosanol-1-yl diphosphate, 21-(halomethyl)-21-tricosanol-1-yl diphosphate.
The phosphohalohydrin compounds in accordance with one of the following formulae may be mentioned among the compounds according to the invention: 
where R2 is an organic or inorganic substituent selected from among the group comprising:
substituents which do not prevent formation of the halohydrin function 
starting from the alkene function 
and halogen X2 in the presence of water;
and substituents for which there is an R2-Oxe2x80x94Y compound which is not reactive towards the halohydrin function of the compound of the formula: 
and selected such that R2-Oxe2x80x94Y may react with the terminal phosphate of this compound (3) in order to obtain the compound (4).
Said compounds according to the invention are advantageously characterized in that n=2 and R1 is CH3.
The compounds according to the invention advantageously additionally comprise at least one group selected from among the group comprising nucleoside derivatives, oligonucleotides, nucleic acids (RNA, DNA), amino acids, peptides, proteins, monosaccharides, oligosaccharides, polysaccharides, fatty acids, simple lipids, complex lipids, folic acid, tetrahydrofolic acid, phosphoric acids, inositol, vitamins, co-enzymes, flavonoids, aldehydes, epoxides and halohydrins.
In particular, the invention provides the phosphohalohydrin compounds of the formula (4) above in which R2 is selected from among the group comprising nucleoside derivatives, oligonucleotides, nucleic acids (RNA, DNA), amino acids, peptides, proteins, monosaccharides, oligosaccharides, polysaccharides, fatty acids, simple lipids, complex lipids, folic acid, tetrahydrofolic acid, phosphoric acids, inositol, vitamins, co-enzymes, flavonoids, phosphohalohydrins of the formula (1), aldehydes, epoxides and halohydrins.
The invention also provides compounds, the structure of which incorporates two or more groups of the formula (1), which may be identical or different, for example monomers, polymers, oligomers or dendrimers, or more generally molecules with two or more phosphorylated branches of the formula (1).
It should be noted that the compounds according to the invention are esters (monoesters or diesters) of phosphoric acid (this term encompassing those acids in which phosphorus is in oxidation state V, namely orthophosphoric acid, pyrophosphoric acid, metaphosphoric acid, triphosphoric acid, other polyphosphoric acids).
The invention provides a process for the production of the compounds according to the invention. According to the invention, halogen X2 is reacted in the presence of water with a starting compound comprising at least one phosphorylated alkene group of the formula: 
Advantageously and according to the invention, a salt formed from said starting compound is reacted in an aqueous or aqueous/alcoholic medium, at neutral pH, at a temperature of below 30xc2x0 C., by mixing with an aqueous solution of the halogen X2. Advantageously and according to the invention, the reaction is performed at atmospheric temperature at a temperature of between 0xc2x0 C. and 25xc2x0 C.
The starting compounds may themselves be obtained from alcohol: 
Advantageously and according to the invention, the starting compound is a salt of the formula: 
The pyrophosphohalohydrin compound of the formula (2) is then obtained.
An example of a complete reaction scheme for obtaining the compound (2) from the alcohol (9) is given below: 
where TsCl is tosyl chloride,
4-DMAP is 4-dimethylaminopyridine,
Bu4N+ is tetrabutylammonium,
(Bu4N+)3HP2O7 is tris(tetra n-butylammonium) hydrogenpyrophosphate,
PP represents the pyrophosphate group.
The reactions which allow compound (6) to be obtained from the alcohol (9) may be performed as described by: DAVISSON V. J. et al.
xe2x80x9cPhosphorylation of Isoprenoid Alcoholsxe2x80x9d J. Org. Chem 1986, 51, 4768-4779, et DAVISSON V. J. et al. xe2x80x9cSynthesis of Allylic and Homoallylic Isoprenoid Pyrophosphatesxe2x80x9d Methods in Enzymology, 1984, 110, 130-144.
Advantageously and according to the invention, the starting compound is a salt of the formula: 
The triphosphohalohydrin compound of the formula (3) is then obtained.
An example of a complete reaction scheme for obtaining the compound (3) from the alcohol (9) is given below: 
where PPP is the triphosphate group,
(Bu4N+)4HP3O10 is tetrakis(tetra-n-butylammonium) hydrogentriphosphate.
The compound (10) is obtained from the alcohol (9) as stated above. The reaction which allows compound (7) to be obtained from the compound (10) may be performed under conditions similar to those described in the publications by DAVISSON V. J. et al or in DAVISSON V. J. et al xe2x80x9cSynthesis of Nucleotide 5xe2x80x2-Diphosphates from (5xe2x80x2-O-Tosyl Nucleosidesxe2x80x9d J. Org. Chem. 1987, 52, 1794-1801.
Advantageously and according to the invention, in a first variant which allows a compound according to the invention of the formula (4) to be obtained, the above-stated production process according to the invention (reaction of X2 in the presence of water with a phosphorylated alkene function) may be performed by using a salt of the following formula as the starting compound: 
where R2 is an organic or inorganic substituent of a nature such as not to prevent formation of the halohydrin function 
starting from the alkene function 
and halogen X2 in the presence of water.
The starting compound (8) may itself be prepared in accordance with one of the following reaction schemes: 
where Ts is tosyl.
The compound (7) may be obtained as stated above starting from the alcohol (9) and the intermediate compound (10). The reaction which allows compound (8) to be obtained from the compound (7) may be performed under conditions similar to those described in the publications by DAVISSON V. J. et al. This scheme may be used when R2-O-Ts is commercially available. 
The intermediate compound (10) may be obtained as stated above starting from the alcohol (9). The reaction which allows compound (8) to be obtained from the compound (7) may be performed under conditions similar to those described in the publications by DAVISSON V. J. et al. This scheme may be used when R2-O-PPP is commercially available. 
where DMF is dimethylformamide,
MeOH is methanol.
This reaction scheme 3 may be performed under conditions similar to those described in D. G. KNORRE et al xe2x80x9cGeneral method for the synthesis of ATP gamma derivativesxe2x80x9d Febs letters, 1976, 70, 105-108.
This reaction scheme 3 cannot be used when R2 comprises a carbodiimide-reactive function (carboxylate, triphosphate etc.). It is, however, advantageous when R2-O-PPP is commercially available.
In the specific case where R2- is itself a halohydrin group of the formula: 
the following reaction scheme may be used: 
This compound (4xe2x80x2) is a particular instance of the compound according to the invention of the formula (4).
It should be noted that, in all these reactions, acetonitrile may be replaced by any other aprotic dipolar solvent (dimethylformamide DMF, dimethyl sulfoxide DMSO etc.).
It should be noted that when preparing compounds (2), (3) and (4xe2x80x2) and in the event that nxe2x89xa02, the intermediate compound (10) may also be replaced by the chloride or bromide compound of the formula: 
where A is chlorine or bromine.
The alcohols (9) are commercially available or may readily be obtained by a well known Grignard reaction between an alkenyl organomagnesium compound and formaldehyde or ethylene oxide.
In a second variant usable in certain cases, a compound of the formula (4) could be prepared by reacting the triphosphate compound according to the invention of the formula (3) starting from a salt soluble in an organic medium, such as a Bu4N+salt, in a subsequent stage with a compound R2-Oxe2x80x94Y, where xe2x80x94Oxe2x80x94Y is a leaving group and R2 is an organic or inorganic substituent selected such that R2-Oxe2x80x94Y is capable of forming, by reaction with the compound (3), the compound according to the invention of the formula: 
In order to be capable of forming the compound of the formula (4) in this manner, the compound R2-Oxe2x80x94Y must in particular not be reactive towards the halohydrin function: 
Moreover, R2-Oxe2x80x94Y must react with the terminal phosphate of compound (3) to form the compound (4).
The reaction of the compound of the formula (3) with R2-Oxe2x80x94Y is a nucleophilic substitution. This reaction is in particular possible and advantageous for R2 selected from among the group comprising alkyls and alkenes. Y is selected such that R2-Oxe2x80x94Y may give rise to the compound (4) by nucleophilic substitution. Y is selected, for example, from among tosyl, brosyl and triflyl.
A compound according to the invention may accordingly be difunctional or multifunctional. The phosphohalohydrin function(s) bring(s) about a specific desired antigenic property towards Txcex39xcex42 lymphocytes, and R2 or the other functional groups of the compound may exhibit other, in particular therapeutic, properties.
In the case of a compound according to the invention having two or more phosphohalohydrin groups of the formula (1), it is sufficient either to start from a starting compound having the corresponding number of phosphorylated alkene groups of the formula (5) and the corresponding chemical structure, or to use the compound of the formula (3) and to react it with an intermediate compound R2-Oxe2x80x94Y having the corresponding number of xe2x80x94Oxe2x80x94Y functions.
The invention also in particular relates to the novel phosphohalohydrin, ester compounds of the formula: 
where X is a halogen selected from among iodine, bromine and chlorine,
R1 is selected from among xe2x80x94CH3 and xe2x80x94CH2xe2x80x94CH3,
Cat+ represents one or more organic or inorganic cation(s) (including the proton), which may be identical or different in the same compound, n is an integer between 2 and 20,
R3- is selected from among:
a halohydrin group of the formula (12),
an epoxide group of the formula: 
an alkene group of the formula: 
m being an integer between 1 and 20.
These compounds (14) are obtained by firstly performing the following initial step: 
Then, the symmetrical diphosphodihalohydrin compound (14a) is obtained as follows: 
The asymmetrical xcex1,xcex2 phosphodiester halohydrin/alkene compound (14b) is obtained as follows: 
The asymmetrical xcex1,xcex2 phosphodiester halohydrin/epoxide compound (14c) is obtained as follows: 
The invention also provides uses of the compounds according to the invention, in particular the compounds of the formula (2), as primate Txcex39xcex42 lymphocyte activators, in particular to activate proliferation and/or cytotoxic activity and/or production of mediator substance(s) for primate Txcex39xcex42 lymphocytes with TCR receptors comprising Vxcex39 and Vxcex42 variable regions.
The invention also provides uses of the compounds according to the invention for the treatment of cells sensitive to primate Txcex39xcex42 lymphocytes in a natural or artificial medium which may contain Txcex39xcex42 lymphocytes, in which medium said cells may be brought into contact with Txcex39xcex42 lymphocytes and which is compatible with the compounds according to the invention (i.e. which is not likely to cause the breakdown thereof, at least under certain treatment conditions).
A xe2x80x9ccell sensitive to Txcex39xcex42 lymphocytesxe2x80x9d is taken to mean any cell subject to the induced effector activity of the Txcex39xcex42 lymphocytes: cell death (cell destruction by Txcex39xcex42 lymphocytes); reception of cytokine released by Txcex39xcex42 lymphocytes (TNF-xcex1, INF-xcex4 . . . ); possibly cellular proliferation induced by Txcex39xcex42 lymphocytes.
The invention accordingly provides a Txcex39xcex42 lymphocyte activation process, in particular a process for activating the proliferation of Txcex39xcex42 lymphocytes and/or the cytotoxic activity of Txcex39xcex42 lymphocytes and/or the production of mediator substance(s) by Txcex39xcex42 lymphocytes, in which process these Txcex39xcex42 lymphocytes are brought into contact with at least one compound according to the invention in a medium which contains Txcex39xcex42 lymphocytes and is compatible with T lymphocyte growth. Advantageously and according to the invention, interleukin, in particular interleukin-2 (IL-2), is introduced into the medium in a proportion suitable to bring about lymphocyte growth in this medium. Indeed, the presence of the lymphocyte growth factor IL-2 is essential to achieve proliferation of the T lymphocytes, among which only the Txcex39xcex42 lymphocytes have been activated by a compound according to the invention. This growth factor must thus be present in the medium for those uses in which proliferation of Txcex39xcex42 lymphocytes is desired. This lymphocyte growth factor may pre-exist in the natural state or be induced or introduced into the medium, simultaneously or not with the incorporation of the compound according to the invention, in the same therapeutic composition or not. Nevertheless, in certain uses in which activation without proliferation of the Txcex39xcex42 lymphocytes is desired (for example induced cytotoxicity), the presence of this growth factor is not helpful.
More specifically, the invention provides uses of the compounds according to the invention for therapeutic purposes for the curative or preventive treatment of pathological conditions producing cells sensitive to primate Txcex39xcex42 lymphocytes in a medium which may contain these Txcex39xcex42 lymphocytes and in which these cells may be brought into contact with the Txcex39xcex42 lymphocytes.
Advantageously and according to the invention, at least one compound according to the invention is used at a concentration in the medium which brings about activation of polyclonal proliferation of Txcex39xcex42 lymphocytes.
This medium may be selected from among human blood, non-human primate blood, human blood extracts, non-human primate blood extracts.
Said medium may be extracorporeal, said activation process according to the invention then being an extracorporeal cellular treatment, in particular applicable in a laboratory, for studying Txcex39xcex42 lymphocytes or the properties thereof, or for diagnostic purposes. The invention also provides a composition for extracorporeal (ex vivo) diagnostics, wherein it comprises at least one compound according to the invention.
Said medium may also be intracorporeal, activation of the Txcex32xcex42 lymphocytes then being of therapeutic use.
More particularly, said medium is the peripheral bloodstream of a primate. The invention accordingly in particular provides a process for the activation of Txcex39xcex42 lymphocytes in the peripheral bloodstream of a primate, in particular humans, into which is administered at least one compound according to the invention in a quantity suitable for activating Txcex39xcex42 lymphocytes. At least one compound according to the invention is thus administered by a general route, in particular parenterally into the peripheral bloodstream.
Said medium may also comprise a cellular site to be treated and at least one compound according to the invention is administered directly in contact with the cellular site to be treated (topical administration).
The invention accordingly in particular provides therapeutic uses of the compounds according to the invention for treating pathological conditions in primates, said conditions belonging to the group comprising cancers, infectious diseases, in particular mycobacterial infections (leprosy, tuberculosis etc.); parasitic conditions (malaria etc.); pathological immunodeficiency syndromes (AIDS etc.). According to the invention, a therapeutic composition is administered which is suitable for releasing into the peripheral bloodstream and/or at a cellular site to be treated a quantity of at least one compound according to the invention capable of activating Txcex39xcex42 lymphocytes. Indeed, it has been demonstrated in general terms in the above-stated prior art that a composition having the property of activating Txcex39xcex42 lymphocytes may advantageously be used for treating these pathological conditions.
As is conventional, throughout the text the terms xe2x80x9ctherapyxe2x80x9d or xe2x80x9ctherapeuticxe2x80x9d encompass not only curative treatment or care, but also preventive treatment (prophylaxis), such as vaccination, together with intracorporeal diagnostics (administration for diagnostic purposes). Indeed, by permitting activation of Txcex39xcex42 lymphocytes, the invention allows immunostimulatory treatments which may be advantageous not only prophylactically by preventing the development of pathogenic cells sensitive to Txcex39xcex42 lymphocytes, but also curatively by inducing destruction of pathogenic cells sensitive to Txcex39xcex42 lymphocytes.
The invention accordingly provides a therapeutic composition comprising at least one compound according to the invention. More specifically, the invention relates to a therapeutic composition comprising a quantity capable of being administered to a primate, in particular in contact with the peripheral bloodstream or topically, of at least one compound according to the invention, in particular for the preventive or curative treatment of the above-stated pathological conditions. A composition according to the invention may be an immunostimulant composition or a vaccine, the compounds according to the invention being antigens which activate Txcex39xcex42 lymphocytes.
Advantageously and according to the invention, the therapeutic composition is characterized in that it moreover comprises a proportion of interleukin, in particular interleukin-2, suitable for bringing about lymphocyte growth in the medium into which it is to be administered.
A therapeutic composition according to the invention may be prepared in a dosage form capable of being administered by a general route, in particular parenterally directly into the peripheral bloodstream of a primate, with at least one compound according to the invention in a quantity suitable to activate Txcex39xcex42 lymphocytes and one or more appropriate excipient(s). Given the very low active concentration of the compounds according to the invention (of the order of 0.1 to 10 nM), such administration may be made without risk of toxicity.
A therapeutic composition according to the invention may also be prepared in a dosage form appropriate for topical administration, directly in contact with the cells sensitive to Txcex39xcex42 lymphocytes.
The dosage form of a therapeutic composition according to the invention is produced in accordance with the selected route of administration using conventional pharmaceutical formulation methods. The quantity and concentration of compound(s) according to the invention and the dosage are determined by reference to known chemotherapeutic methods for the diseases to be treated, taking account of the bioactivity of the compounds according to the invention towards Txcex39xcex42 lymphocytes, the individual to be treated, the disease in question and the desired biological effects.
Advantageously and according to the invention, in the case of a bioactive compound at a concentration of between 1 nM and 10 nM, the quantity of compound(s) according to the invention administered by a general route is between 0.1 xcexcg and 100 xcexcg, in particular between 1 xcexcg and 10 xcexcg, per kilogram of patient body weight.
It has moreover been demonstrated in vitro that the compounds according to the invention exhibit no general toxicity even at concentrations of up to 100 xcexcM, i.e. of the order of 105 times the bioactive concentration. Furthermore, it is known that the biochemical class of molecules to which the compounds according to the invention belong (phosphoesters) comprises a family of metabolic compounds found in any living cell. The compounds according to the invention thus exhibit no toxic effects other than those induced by the bioactivity thereof upon Txcex39xcex42 lymphocytes.
Moreover, certain compounds according to the invention have a sufficiently low molecular weight (in particular below 500) to be compatible with the elimination thereof via the kidneys and urine.
One example formulation of an injectable therapeutic composition according to the invention for a primate weighing 1 kg is as follows: 5 xcexcg of 3-(iodomethyl)-3-butanol-1-yl diphosphate (IHPP) diluted in 0.5 ml of sterile phosphate buffer at pH 7 and adjusted to 37xc2x0 C.
In this manner, 5 xcexcg of IHPP (compound of the formula (2)) are administered per 1 kg of animal body weight, corresponding to a concentration in the circulating blood such as to be greater than the bioactive concentration of IHPP (a concentration of 10 nM of IHPP corresponding to approx. 5 ng/ml).
It should be noted that the majority of the excipients or other conventionally used pharmaceutically acceptable additives are chemically compatible with the compounds according to the invention.
A therapeutic composition according to the invention may also advantageously comprise one or more other active ingredient(s), in particular to bring about a synergistic effect. In particular, a compound according to the invention may act as a vaccine adjuvant. The therapeutic vaccine composition according to the invention then comprises a known vaccine composition to which is added a quantity of compound(s) according to the invention capable of activating the Txcex39xcex42 lymphocytes which will not only be able to exert their anti-infective activity directly, but will also be able to activate the T lymphocytes which effect the conventional vaccine response.
A therapeutic composition according to the invention may also itself incorporate primate Txcex39xcex42 lymphocytes in a culture in a medium compatible with T lymphocyte growth. It may then be used for treating primates, or more generally vertebrates with which administration of primate Txcex39xcex42 lymphocytes may be performed under conditions of immune compatibility towards said primate Txcex39xcex42 lymphocytes. Such a composition according to the invention may be administered by a general route, or even by a topical route, in contact with target pathogenic cells, sensitive to said primate Txcex39xcex42 lymphocytes.
The invention also provides the use of at least one compound according to the invention for the production of a therapeutic composition according to the invention. More particularly, the invention relates to the use of at least one compound according to the invention for the production of a therapeutic composition intended for the preventive or curative treatment of a pathological condition of humans or vertebrates which produces cells sensitive to primate Txcex39xcex42 lymphocytes, in particular a pathological condition selected from the group comprising cancers, infectious diseases, parasitic conditions and pathological immunodeficiency syndromes. To this end, the invention also provides the use of at least one compound according to the invention for the production of a therapeutic composition intended to be administered, in particular in contact with the peripheral bloodstream or by a topical route, to a primate, in particular to humans, for the preventive or curative treatment of a pathological condition as mentioned above.
The invention also provides a process for the production of a composition, in particular a therapeutic composition, according to the invention having the characteristic of activating Txcex39xcex42 lymphocytes, in which process at least one compound according to the invention is used.
The invention also relates to a process for the production of a therapeutic composition intended for the preventive or curative treatment of a pathological condition of humans or vertebrates which produces cells sensitive to primate Txcex39xcex42 lymphocytes, in which process at least one compound according to the invention is used. The invention in particular relates to a process for the production of a therapeutic composition intended to be administered, in particular in contact with the peripheral bloodstream or by a topical route, to a primate, for the preventive or curative treatment of a pathological condition which produces cells sensitive to Txcex39xcex42 lymphocytes, in particular a pathological condition belonging to the group stated above, in which process at least one compound according to the invention is used.
Advantageously and according to the invention, in a production process according to the invention, at least one compound according to the invention is brought into contact with a medium which contains/Txcex39xcex42 lymphocytes, and is compatible with T lymphocyte growth, in a quantity suitable to activate these Txcex39xcex42 lymphocytes in this medium. Advantageously and according to the invention, said medium comprises a substance selected from among primate blood and primate blood extracts. A therapeutic composition containing activated Txcex39xcex42 lymphocytes is then obtained so allowing a cellular therapeutic approach to be performed.
It should be noted that the compounds according to the invention are halogenated and, for this reason alone, cannot correspond to natural phosphoantigens, in particular to the molecules known as Tubag1, Tubag2, Tubag3 and Tubag4 obtained as described in WO 95/20673. It is in any event possible to demonstrate for example that these natural phosphoantigens are broken down by the bromine water used for the chemical production of the phosphobromohydrins according to the invention. The compounds according to the invention are thus not natural antigens, but are synthetic antigens which activate Txcex39xcex42 lymphocytes at concentrations of the same order and with an efficiency similar to or even greater than that of natural antigens.
It should also be noted that, contrary to the prior art as illustrated by U.S. Pat. No. 5,639,653, which considered that the presence of an alkyl or alkene group was essential to activate human Txcex39xcex42 lymphocytes, the inventors have observed that by destroying the alkene bond with the addition of halogen, an element absent from natural biological compounds, the Txcex39xcex42 lymphocytes are activated extremely strongly and at very low concentration. In particular, it may be observed that the effect may even exceed that of phosphoantigens of natural origin.